Visualizing the First Steps of Human Double-Strand Break Repair on a Crowded DNA Track

Homologous recombination (HR) is a universally conserved and accurate DNA double strand break (DSB) repair pathway. In humans, HR is initiated by the Mre11/Rad50/Nbs1 (MRN) complex, which rapidly localized to the DSB and recruits nucleases that process the free DNA ends for downstream recombination. This process must occur on chromatin, but little is known about the first steps of HR on a nucleosome coated DNA. Here, we use high-throughput DNA curtains to visualize the first steps of human HR. We show that MRN scans DNA via one-dimensional facilitated diffusion. Remarkably, MRN uses its many DNA-binding modes to bypass nucleosomes and other roadblocks as it searches for DSBs. Next, MRN recruits Exonuclease 1 (Exo1), which uses its 5’→3’ nuclease activity to process the free DNA ends. Exo1 is a processive enzyme that can digest thousands of base pairs of DNA in a single resection event. However, RPA and other single-stranded DNA binding proteins (SSBs) inhibit Exo1 by stripping the nuclease from DNA. RPA inhibition is not species-specific, and requires at least three of its many DNA-binding domains. Strikingly, SOSS1—a recently identified mammalian SSB that is required for DSB repair in human cells—supports long-range Exo1 nuclease activity. Our results provide an integrated model for how DSB repair is initiated on a crowded DNA track and how single-stranded DNA-binding proteins regulate the first steps of human DSB repair.